Acute otitis media (AOM) is the most common childhood infection in the United States, affecting over 60% of infants during their first year of life, causing significant pain and transient hearing loss. If left untreated, a prolonged infection can lead to serious complications, including delays in speech and language development and even permanent hearing impairment. Streptococcus pneumoniae (pneumococcal) is the most commonly isolated etiologic agent of AOM, and is also the species responsible for most post-infectious complications. The pneumococcal capsular conjugate vaccine is effective in preventing invasive disease, but has had little impact on total episodes of AOM due to increased rates of infection with non-vaccine serotypes. This fact, coupled with the global emergence of antibiotic-resistant pneumococcal strains, emphasizes the urgent need for continued efforts in developing effective preventive and therapeutic agents against pneumococcal disease. Previous work has identified a protein-lipid complex from human milk (HAMLET) that effectively kills S. pneumoniae, using a bactericidal mechanism that is separate from common antibiotics and is not susceptible to bacterial resistance development. This mechanism is specific, and treatment with HAMLET has been shown to be effective in significantly reducing in vivo nasopharyngeal colonization in a mouse model, revealing great promise for future use in treating otitis media in children. The objectives of this proposal are to develop a greater understanding of the mechanism of HAMLET-induced death in S. pneumoniae, and to assess the utility of HAMLET as a therapeutic agent. It is hypothesized that HAMLET targets pneumococcal components and genetic machinery specifically used for cell suicide, and that this activity can be exploited to prevent and treat pneumococcal colonization and otitis media. The specific aims of this proposal have been designed to directly test these hypotheses. Studies proposed in Aim 1 will characterize the mechanism of HAMLET-induced death in the pneumococcus. First, the receptor structure for HAMLET will be elucidated by testing various preparations of choline-containing pneumococcal surface components for their ability to inhibit HAMLET-induced death and to interact with HAMLET in solid-phase and in vitro binding assays. Secondly, a signature-tagged transposon mutation library will be screened to identify ion transporters or other pneumococcal components critical for HAMLET's activity. In Aim 2, the proposed studies will assess the ability of HAMLET to prevent and eradicate nasopharyngeal colonization and prevent otitis media in vivo using the chinchilla, the gold standard animal model of AOM. Results from these studies will determine the utility of HAMLET for future treatment studies in children and also have great potential to lead to the development of better preventive and therapeutic agents against pneumococcal colonization and AOM, with less risk for resistance development.